Cloning vectors for streptomyces and use thereof in macrolide antibiotic production

ABSTRACT

DNA cloning shuttle vectors, including a cosmid shuttle vector, for E. coli and Streptomyces are disclosed. Specifically, disclosed shuttle vectors pAL7002 (NRRL B-18055) and pNJl (NRRL B-18054) contain an E. coli origin of replication, Streptomyces replication functions, and antibiotic resistance markers for both E. coli and Streptomyces. In addition, pNJl contains a cos sequence. Novel 2-norerythromycin antibiotics A, B, C, and D, which were produced in a strain Streptomyces erythreus 12693-240 (NRRL B-18053) transformed by pNJl bearing DNA from Streptomyces antibioticus, are also disclosed. The present invention also provides a method for producing novel antibiotics. This method for antibiotic production is applied to the transformation of a blocked mutant of S. erythreus with genomic DNA from S. antibioticus but may be more broadly applied to genes to antibiotic-producing strains transformed into cells which are blocked in the pathway for production of a different antibiotic.

BACKGROUND

The present invention relates in general to shuttle vectors for use withE. coli and Streptomyces and cloning of antibiotic genes using thesevectors. In particular, the present invention relates to vectors pNJland pAL7002 and to production of 2-norerythromycin antibiotics employingthese vectors.

Cloning vectors, called E. coli-Streptomyces shuttle vectors, may beconstructed which contain origins of replication and antibioticresistance markers for both E. coli and in Streptomyces. Nakatsukasa etal., U.S. Pat. No. 4,513,085; Fayerman et al., U.K. Patent ApplicationNo. 2,107,716; Fayerman et al., U.K. Patent Application No. 2,107,717;and Hershberger et al., U.K. Patent Application No. 2,118,947. However,because Streptomyces strains are used to produce most of the clinicallyimportant antibiotics, it is desirable to obtain stable vectors whichreplicate and are selectable in E. coli, where recombinant manipulationsare relatively easy to perform, and which replicate and are selectablein Streptomyces, wherein recombinantly-manipulated genes are expressed.Stability is particularly important inasmuch as some such shuttlevectors may be unstable, i.e. may be lost from their host cell alongwith the cloned gene they carry. See e.g. Matsushima et al.,Bio/Technology, 4, 229-232 (1986).

Because cosmid vectors combine the opportunity to make use of the widevariety of features of plasmid vectors, including selection markers, andthe large cloning capacity of phage vectors, they are particularlydesirable. E. coli-Streptomyces cosmid shuttle vectors may beconstructed to take advantage of these features. Matsushima et al.,supra. Cosmid shuttle vectors which are stable with application ofselection pressure are also desirable.

Erythromycin is a macrolide antibiotic product of a biosynthetic pathwayin Streptomyces erythreus which is believed to have about thirty steps.Cosmid shuttle vectors carrying genes for antibiotic production may beused to explore biosynthesis of hybrid antibiotics. Matsushima et al.,supra. Attempts to assay for the production of erythromycin may involvethe use of S. erythreus strains having no detectable antibiotic activitywhich are blocked in the biosynthesis of erythromycin, such as eryAmutants which are blocked in the formation of 6-deoxyerythronolide B, anunmodified lactone. Matsushima et al., supra. Restoration of antibioticfunction in such strains after transformation with a vector carryinggenetic material to be tested for the production of erythromycinindicates antibiotic function is restored by the vector-borne geneticmaterial. However, no suggestion has been made that such a techniquemight be employed to obtain hitherto unreported analogs of erythromycinsuch as norerythromycins.

SUMMARY OF THE INVENTION

A DNA cloning vector according to the present invention includes afragment of plasmid pAL7002 having a functional E. coli origin ofreplication, functional determinants for replication in Streptomyces, amarker selectable in E. coli, a marker selectable in Streptomyces. Morespecifically, such a vector is a plasmid pAL7002 isolated from E. coliHB101/pAL7002 NRRL B- 18055, has a molecular size of about 6.2 kb, has athiostrepton resistance segment, has an ampicillin resistance segment,has a unique HindIII site, has a unique PstI site, has a unique PvuIIsite, has a unique EcoRI site, has a unique BglII site, and has two SmaIsites.

Another DNA cloning vector according to the present invention is stableunder selection in S. erythreus and includes a fragment of plasmid pNJlhaving a functional E. coli origin of replication, functionaldeterminants for replication in Streptomyces, a marker selectable in E.coli, a marker selectable in Streptomyces, and a cos segment. Morespecifically, such a vector is a plasmid pNJl isolated from E. coliHB101/pNJ1 NRRL B-18054, has a molecular size of about 8.8 kb, has athiostrepton resistance segment, has an ampicillin resistance segment,has a unique HindIII site, has a unique PstI site, has a unique PvuIIsite, has a unique EcoRI site, has a unique BglII site, and has two SmaIsites.

A strain of S. erythreus according to the present invention has all ofthe identifying characteristics of S. erythreus 12693-240 NRRL B-18053.More specifically, a presently preferred embodiment of this strain is abiologically pure culture of S. erythreus producing upon cultivation inan aqueous medium containing assimilable sources of nitrogen and carbona member from the group consisting of: 2-norerythromycin A;2-norerythromycin B; 2norerythromycin C; and 2-norerythromycin D orpharmaceutically acceptable salts of these members.

A process for producing a 2-norerythromycin A antibiotic according tothe present invention includes the step of cultivating S. erythreus12693-240 NRRL B-18053 in an aqueous medium in the presence ofassimilable sources of nitrogen and carbon.

A process for producing an erythromycin antibiotic according to thepresent invention includes the step of cultivating S. erythreus12693-240 NRRL B-18053 in an aqueous medium in the presence ofassimilable sources of nitrogen and carbon.

The present invention provides the hitherto unreported compound2-norerythromycin A or a pharmaceutically acceptable salt thereof whichmay be obtained by means well known to those skilled in the art ofobtaining pharmaceutically acceptable salts of erythromycin. This2-norerythromycin A is preferably the 2-norerythromycin A effective ininhibiting the growth of bacteria and in substantially pure form whichhas a mass spectrum, FAB positive ion MH⁺ m/z of 720.

The present invention also provides the hitherto unreported compound2-norerythromycin B or a pharmaceutically acceptable salt thereof whichmay be obtained by means well known to those skilled in the art ofobtaining pharmaceutically acceptable salts of erythromycin. This2-norerythromycin B is preferably one effective in inhibiting the growthof bacteria and one which in substantially pure form has a massspectrum, FAB positive ion MH⁺ m/z of 704.

The present invention further provides the hitherto unreported compound2-norerythromycin C or a pharmaceutically acceptable salt thereof and ispreferably the 2-norerythromycin C which is effective in inhibiting thegrowth of bacteria and which in substantially pure form has an ¹ H-NMRspectrum comprising the partial spectrum as set forth in Table 3.

In addition, the present invention provides the hitherto unreportedcompound 2-norerythromycin D or a pharmaceutically acceptable saltthereof. Preferably, this 2-norerythromycin D is one which is effectivein inhibiting the growth of bacteria and which in substantially pureform has an ¹ H-NMR spectrum as set forth in Table 1 and has a ¹³ C-NMRspectrum as set forth in Table 2.

An antibiotic composition according to the present invention includes adiluent or carrier, such formulations being readily obtainable by thoseskilled in the art of preparing formulations for erythromycins,compatible with antibiotic activity and an antibiotic selected from thegroup consisting of 2-norerythromycin A, 2-norerythromycin B,2-norerythromycin C, and 2-norerythromycin D or pharmaceuticallyacceptable salts of these antibiotics.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a partial restriction map of a vector pAL7002 according to thepresent invention;

FIG. 2 is a partial restriction map of a vector pNJl according to thepresent invention;

FIG. 3 is a composite diagrammatic representation of the structure of2-norerythromycins A, B, C, and D; and

FIG. 4 is a diagrammatic representation of the association betweendifferences in the ¹³ C NMR spectra of erythromycin B or C and2-norerythromycin D and the structures thereof.

DETAILED DESCRIPTION

The present invention includes selectable DNA cloning vectors comprisinga fragment of plasmid pJVl containing determinants for replication inStreptomyces, a fragment of DNA encoding selection for antibioticresistance in Streptomyces, a functional origin ofreplication-containing and antibiotic resistance-conferring restrictionfragment of a plasmid which is functional in E. coli and the cossequence which contains the cohesive ends from bacteriophage lambda.Other selectable DNA cloning vectors according to the present inventioninclude all of the above but lack the cos sequence. The vectors of thisinvention are functional in Streptomyces erythreus and other hoststrains. The vectors are useful because they are small, easilyhandleable, and may be introduced into a wide variety of host strainsincluding Streptomyces erythreus and E. coli.

Because Streptomyces produce over half of the clinically importantantibiotics, it is desirable to develop vectors which could be used fortransfer of DNA between different species or between strains ofStreptomyces in order to increase yields of existing antibiotics or inorder to produce new antibiotics or their derivatives. Vectors accordingto the present invention are useful for these purposes. The vectorsaccording to the present invention which contain the cos sequence areparticularly useful because they may be employed to clone large DNAsegments from different sources which may be up to 35 kilobases inlength and which may contain twenty or more contiguous genes.

The vectors of the present invention are constructed by step-wiseligation of various DNA fragments in vitro and subsequent transformationof sensitive host cells. The fragments which contain the components ofthe vectors are themselves obtainable from Streptomyces or E. coliplasmids which are either commercially available or which may beobtained from commercial sources.

Reagents & Enzymes

Media for the growth of bacteria were purchased from Difco, Detroit,Mich., or Gibco, Long Island, N.Y. Restriction enzyme, calf intestinalalkaline phosphatase (CIAP) and T4 DNA ligase were purchased fromBethesda Research Laboratories, (BRL), Gaithersburg, Md., New EnglandBiolabs, Beverley, Mass., or Boehringer Mannheim, Indianapolis, Ind. TheGigapack™ Kit containing reagents for the in vitro packaging ofbacteriophage lambda DNA was purchased from Vector Cloning Systems, SanDiego, Calif. Agarose was purchased from Bio Rad, Richmond, Calif. Eggwhite lysozyme (3X crystallized) was obtained from Sigma, St. Louis, Mo.Thiostrepton was obtained from E. R. Squibb and Sons, Princeton, N.J.

Host Cell Cultures, DNA Sources and Vectors

E coli K12 strains JM83 and HB101 were obtained from BRL. Streptomyceslividans 66, was obtained from the John Innes Institute, Norwich, U.K.S. lividans 66 carrying plasmid pIJ704 was obtained from E. Katz atGeorgetown University. Streptomyces phaeochromogenes NRRL B-3559carrying plasmid pJVl was obtained from the Agricultural CultureCollection (NRRL), Peoria, Ill., under the accession number NRRL B-3559.

E. coli vector pUC9 and bacteriophage lambda were purchased fromBethesda Research Laboratories or PL Laboratories, Milwaukee, Wis.Plasmid pJB8 was obtained from Mill Hill Laboratories, London, U.K. andis also available from the American Type Culture Collection, Rockville,Md., under the accession No. 37074.

General Methods

Restriction enzymes and T4 DNA ligase were used according to suppliers'instructions. Standard procedures for the growth and transformation ofE. coli [Maniatis, et al., "Molecular Cloning, A Laboratory Manual,"Cold Spring Harbor, N.Y. (1982)], growth and transformation ofStreptomyces lividans [Bibb et al., "Developments in StreptomycesCloning," in Experimental Manipulation of Gene Expression, Inouye ed.,Academic Press, New York, N.Y., 53-87 (1983)], analysis of DNA onagarose gels [Maniatis et al., "Molecular Cloning, A Laboratory Manual,"Cold Spring Harbor, N.Y. (1982)], and labeling of DNA by nicktranslation [Maniatis et al., "Molecular Cloning, A Laboratory Manual,"Cold Spring Harbor, N.Y. (1982)]were used. Plasmids were isolated fromE. coli and purified using published procedures [Maniatis et al.,"Molecular Cloning, A Laboratory Manual," Cold Spring Harbor, N.Y.(1982)].

The present invention is described in more detail in the followingexamples.

In Example 1, the gene determining thiostrepton resistance (tsr) wassubcloned from the plasmid pIJ704 into E. coli vectors such as pUC9 toyield the vector pUC9tsr. In Example 2, plasmid pJVl is isolated fromStreptomyces phaeochromogenes and a fragment of pJVl that carriesStreptomyces replication functions is subcloned into the plasmid pUC9tsryielding the plasmid pAL7002.

Example 3 describes the subcloning of a fragment of plasmid pAL7002(which carries Streptomyces replication functions and the tsr gene) intoa fragment of plasmid pJB8 (which carries E. coli replication functions,the amp gene for selection of ampicillin resistant transformants in E.coli and the sequence cos which are the cohesive ends of thebacteriophage lambda) to yield the plasmid pNJl.

In Example 5, a genomic library of Streptomyces antibioticus strain ATCC11891 is constructed in pNJl and is introduced into E. coli.

In Example 6, plasmid DNA from E. coli carrying the genomic libraries ofStreptomyces antibioticus ATCC 11891 is prepared and is transformed intoStreptomyces erythreus strain 9EI41, an antibiotic non-producer,employing selection for resistance to the antibiotic thiostrepton.Thiostrepton resistant transformants are tested in Example 6 for abilityto produce an antibiotic and an antibiotic producer, S. erythreus strain12693-240, is detected.

Example 7 sets forth the fermentation of S. erythreus 12693-240, theextraction of the fermentation beer, and the identification of products.Also in Example 7, the compounds 2-norerythromycins A, B, C, and D areidentified from the fermentation beer of S. erythreus 12693-240.

EXAMPLE 1 Construction of pUC9-tsr

The plasmid pUC9 was cut with the restriction enzyme BamHI understandard conditions and the DNA was recovered after precipitation inethanol. This procedure renders the cut DNA free of the enzyme andbuffer. The plasmid pIJ704, isolated from Streptomyces lividans 66 bythe isolation procedure described in Example 2, was cut with the enzymeBClI and the resulting fragments were separated by agarose gelelectrophoresis and were visualized under ultraviolet light afterstaining with ethidium bromide. A band corresponding to DNA of sizeapproximately 1 kb was identified and removed from the gel byelectroelution wherein the DNA fragments to be recovered were removedfrom the gel and placed in a dialysis sac containing the gel buffer. Thesac was sealed and placed on a horizontal gel apparatus containingbuffer and was subjected to electrophoresis for a period long enough tovisualize the migration of the UV-fluorescent material from the gelslice into the surrounding buffer.

The buffer was recovered, and after purification by phenol-chloroformextraction and concentration by ethanol precipitation, approximately 2μg of the 1 kb BClI fragment was mixed with 0.5 μg of BamHI-cut pUC9 ina standard buffer for T4 DNA ligase and 2 units of T4 DNA ligase wasadded in a total volume not exceeding 40 μl. The mixture was incubatedat 14° C. overnight. Approximately 100 ng of the mixture was used in thetransformation of competent E. coli JM83 cells by the procedure ofHanahan et al., J. Mol. Biol., 166, 557-580 (1983) and employingselection for ampicillin resistance on LB-agar plates containing 10μg/ml of Xgal. White colonies, the color of which indicated the presenceof an insertion of DNA into pUC9, were selected, picked, and examinedfor their plasmid content. A strain, designated E. coli JM83/pUC9-tsr,carrying a plasmid of approximately 3.6 Kb which was not cut by theenzyme BamHI, was retained. The plasmid was designated pUC9-tsr. Furthercharacterization of this plasmid revealed that it contained therestriction sites as reported for the tsr gene of pIJ704 in Thompson, etal., Gene, 20, 51-62 (1982).

EXAMPLE 2 Construction of Vector pAL7002

A plasmid designated pJVl, as described in Doull et al., FEMS Microbiol.Lett., 16, 340-352 (1983) was isolated from mycelia of 4 to 6 day oldcultures of Streptomyces phaeochromogenes (NRRL B-355)) using thefollowing procedure followed throughout this application for isolatingplasmids from Steptomyces. Cells were harvested by centrifugation,washed once in 10% sucrose, were resuspended in 1/50th volume ofmodified P medium (MgCl₂ and CaCl₂ each reduced to 0.005 M) [Okanishi etal., J. Gen. Microbiol., 80, 389-400 (1974)]containing 5 mg/ml oflysozyme. The preparation was incubated at 32° C. until protoplastformation was observed. Two times the volume of lysing solution (0.3 NNaOH, 2% SDS, 0.05M EDTA) was added to the preparation. The mixture wasincubated at 50° C. for 30 minutes. Two times the volume of 4 M sodiumactetate, (pH 4.8) was added to the preparation and the mixture wasvigorously shaken for 1 minute. The preparation was allowed to sit onice for 30 minutes and was then centrifuged at 15,000 rpm in arefrigerated centrifuge. The supernatant was recovered and wasconcentrated by precipitation after addition of 1 volume of isopropanoland incubation on ice for 10 minutes. The precipitate was resuspended inTE buffer and plasmid DNA was recovered after centrifugation of thepreparation in cesium chloride-ethidium bromide density gradients.

The plasmid pJVl was cut with the enzyme SmaI and the fragmentsgenerated were subjected to agarose gel electrophoresis, phenolextraction and ethanol precipitation. Approximately 2 μg of thispreparation was mixed with 0.5 μg of SmaI-cut, ethanol precipitatedpUC9-tsr and ligated in the mannner described in Example 1. One-half ofthe preparation was then used to transform protoplasts of Streptomyceslividans 66 according to the procedure of Bibb et al., "Developments inStreptomyces Cloning," supra, employing selection for thiostreptonresistance. A number of these clones that appeared were examined fortheir plasmid content. A strain, designated Streptomyces lividans66/pAL7002, that harbored a plasmid of approximately 6.2 kb was kept.The plasmid, designated pAL7002, was isolated. Plasmid pAL7002 may beisolated from E. coli HB101/pAL7002 which was deposited on Mar. 11, 1986with the Agricultural Research Culture Collection (NRRL), Peoria, Ill.,as deposit No. NRRL B-18055. A partial restriction and genetic map ofthe plasmid is shown in FIG. 1.

EXAMPLE 3 Construction of Vector pNJl

When the ˜1.05 kb BClI thiostrepton resistance-determining fragment isinserted into the BamHI site of plasmid pUC9 and the ˜2.7 kb fragment ofpJVl is subsequently inserted into the SmaI site of the compositepUC9-tsr vector, the vector pAL7002 is produced. The proximity of thethiostrepton resistance determinant to the replication-functionalsegment in pAL7002 allows them to be subcloned into other vectors as asingle HindIII-EcoRI restriction fragment of ˜3.8 kb. As, for example,when the ˜3.8 kb HindIII-EcoRI is subcloned into HindIII and EcoRIcutvector pJB8, the resulting vector pNJl is produced.

Approximately 5 μg of pAL7002 was cut with the restriction enzymeHindIII using standard conditions. The DNA was then treated with theenzyme calf intestinal alkaline phosphatase (CIAP) to remove theterminal phosphate from the free ends of the cut plasmid. The DNA wasthen phenol extracted, precipitated and redissolved and then subjectedto restriction with the enzyme EcoRI for 2 hours. After treatment, theDNA was phenol extracted and precipitated as described in Example 1 toform a first preparation. Similarly, 5 μg of pJB8 was first cut with theenzyme EcoRI, treated with enzyme CIAP, treated with HindIII andrecovered after phenol extraction and precipitation to form a secondpreparation. One microgram of each preparation was mixed together andligated as described. The resulting preparation was then transformedinto competent E. coli HB101 cells employing selection for ampicillinresistance. Plasmid DNA was examined from a number of colonies. Astrain, designated E. coli HB101/pNJl, carrying a plasmid ofapproximately 8.8 kb was kept. The plasmid was designated pNJl. PlasmidpNJl may be isolated from E. coli HB101/ pNJl which was deposited onMar. 11, 1986, with the Agricultural Research Culture Collection (NRRL),Peoria, Ill., under the accession No. B-18054. A partial restriction mapof the plasmid is shown in FIG. 2.

EXAMPLE 4 Construction of S. erythreus/pAL7002 and S. erythreus/pJN1

Plasmids pAL7002 or pNJl were transformed into protoplasts ofStreptomyces erythreus according to the following procedure.

In a general procedure for growth of Streptomyces and conversion toprotoplasts, spores, or vegetative mycelia of Streptomyces erythreuswere inoculated into 50 ml of SGGP medium (0.4% Tryptone, 0.4% yeastextract, 0.05% MgSO₄, 1% glucose. 0.2% glycine, 0.01 M potassiumphosphate buffer, pH7.0) and were incubated at 28° C. with shaking at200 rpm for a period of 4 to 6 days. The cells were then harvested bycentrifugation, were washed once in P medium [Okanishi et al.,supra,]and were resuspended in P medium containing lysozyme at a finalconcentration of 5 mg/ml. The suspension was incubated at 32° C. fortime sufficient (generally 15 to 60 min) to allow protoplast formationas monitored by examination of samples under a microscope. Protoplastswere collected by low speed centrifugation, washed free of the lysozymein P medium and were resuspended at a final density of >10¹⁰ /ml.Protoplasts were either used directly for transformation or were storedat -70° C. after addition of DMSO to a final concentration of 4%.

In the transformation of S. erythreus protoplasts, 0.5-5 μg of DNA in TEbuffer [Maniatis et al., "Molecular Cloning, A Laboratory Manual," ColdSpring Harbor, N.Y., (1982)]was mixed with 10 μg of heparin, was chilledon ice for 20 minutes and then was brought to room temperature where allsubsequent steps take place. Protoplasts at a concentration of about 10⁹/ml were added to the DNA followed after 1 minute by addition of 25%polyethylene glycol 3350 in PT medium (10.3% sucrose, 0.025% K₂ SO₄,0.025M TES buffer, pH 7.2, 0.025 M each of CaCl₂ and MgCl₂ and traceelements as described in Bibb et al., Nature, 274, 398-400 (1978)).After 2 minutes standing, undiluted aliquots were spread with the aid ofa plastic rod over the surface of predried R3M agar media [2.2% agar,0.4% each of Tryptone, Casamino acids and Yeast Extract, 0.025MTris-HCl, pH 7.2, 0.05% KH₂ PO₄, 0.05M each of CaCl₂ and MgCl₂, 0.0025MNaOH, 0.025% K₂ SO₄, 1% glucose, 10.3% sucrose, and trace elements as inBibb et al., Nature, 274, 398-400 (1978)]. Plates were incubated at 28°C. for 18 to 36 hours until regeneration was evident. For selection ofthiostrepton resistant (Thio®)transformants, the plates were overlayedwith 5 ml of semisolid Trypticase Soy Broth containing 0.6% agar and 40μg/ml of thiostrepton, and were incubated at 32° C. until coloniesappeared. The ability of colonies to grow on a medium containingthiostrepton indicated that they were composed of transformantscontaining the Thio® segment.

EXAMPLE 5 Construction of a Genomic Library of Streptomyces antibioticusATCC 11891 in pNJl and Introduction into E. coli

Genomic DNA from S. antibioticus strain, available from the Americantype Culture Collection as No. ATCC 11891 was prepared from protoplastsof Streptomyces antibiotics, prepared as described in Example 4, whichwere lysed with a solution of SDS or sarkosyl. The lysate is thendeproteinated by repeated treatments with phenol-chloroform, followed byconcentration by ethanol precipitation. The dissolved precipitated istreated with ribonuclease, then re-extracted with phenol-chloroform.After precipitation with ethanol, the dissolved material is usable forcloning. The dissolved material was partially cut with the enzyme Sau3Alto yield fragments ranging in size from ˜1 kb to >40 kb.

The DNA fragments were treated with the enzyme CIAP, were extracted withphenol, and were precipitated with ethanol (preparation A). Plasmid pNJlwas digested with EcoRI, was phenol extracted, was treated with CIAP,was phenol extracted again, was treated with the restriction enzymeBglII, was phenol extracted and was precipitated (preparation B).Similarly, a second sample of pNJl was digested with the restrictionenzyme HindIII, was treated with CIAP, and then was digested with BglII(preparation C). A mixture consisting of 0.5μg of preparation B, 0.5 μgof preparation C and 5 μg of preparation A was prepared in ligationbuffer and was treated with T4 DNA Ligase at 14° C. overnight. Thematerial was then subjected to in vitro packaging in bacteriophagelambda using the Gigapack™ kit employing the protocols obtained from thesupplier.

After packaging was completed, the preparation was mixed with E. coliHB101 cells in 0.01 M MgSO₄ to permit adsorption of the phage onto thecells and allowed to stand at room temperature for 20 minutes. The cellswere then transferred to L Broth and allowed to grow for a period of 1hour before addition of ampicillin to the medium to a finalconcentration of 50 μg/ml. The culture was incubated overnight andplasmid DNA was extracted from the preparation.

EXAMPLE 6 Construction of Streptomyces erythreus 12693-240

The mixed plasmid preparation of pNJl carrying components of the genomiclibrary of Streptomyces antibioticus ATCC 11891, anoleandomycin-producing strain, prepared as described in Example 5, wasisolated from E. coli and transformed into protoplasts of Streptomyceserythreus strain 9EI41, an eryA strain, employing selection forthiostrepton resistance as described in Example 4. Individualtransformants that arose were tested for the production of one or moresubstances which either killed or inhibited the growth of a thiostreptonresistant culture of Staphylococcus aureus in an agar plug test asfollows. Transformants, obtained by picking isolates which grew in thepresence of thiostrepton, were grown on SLM-3 agar medium (1%cornstarch, 0.5% cornsteep liquor, 0.00012% FeSo₄, 0.3% CaCO₃, 1.5%agar, pH adjusted to 7.0) containing thiostrepton at 2-5 μg/ml tomaintain selection for the presence of vectors containing the gene forresistance to thiostrepton, for 7-10 days at 32° C. A small plug of theagar, taken from the area of dense growth was then placed on the surfaceof an agar medium seeded with a suspension of thiostrepton resistantStaphylococcus aureus cells. The medium was incubated overnight at 37°C. If an antibiotic was produced by the Streptomyces culture, a zone ofinhibition of the S. aureus was seen to surround the agar plug.

One isolate, designated Streptomyces erythreus 12693-240, exhibited thecharacteristic of, when grown in the presence of thiostrepton, producingone or more substances that killed or inhibited the growth ofthiostrepton-resistant Staphylococcus aureus cells. S. erythreus12693-240 was deposited on Mar. 11, 1986, and made part of the permanentculture collection of the Agricultural Research Culture Collection(NRRL) Peoria, Ill., under the accession number NRRL B-18053.

EXAMPLE 7 Antibiotics from S. erythreus 12693-240

Streptomyces cells of strain 12693-240 were inoculated into 500 ml ofSCM medium (1.5% soluble starch, thiostrepton at 2 μg/ml and grown for 3to 6 days at 32° C. thiostrepton at 2 μg/ml and grown for 3 to 6 days at32° C. The entire culture was then inoculated into 10 liters of freshSCM medium containing thiostrepton at 2 μg/ml and 5% soy bean oil in afermenter. The fermenter was operated for a period of 7 days at 32° C.maintaining constant aeration and pH at 7.0.

At harvest, the fermentation beer was adjusted to pH 9.2 with 5N KOH,centrifuged and the supernatant beer was extracted sequentially with twohalf-volumes of ethyl acetate. The combined ethyl acetate extracts wereextracted with two half-volumes of pH 4.8, 2% citric acid-sodium citrateaqueous buffer. The combined aqueous layers were adjusted to pH 9.2 with5 N KOH and extracted with 2 halfvolumes of ethyl acetate. The combinedethyl acetate extracts were concentrated to yield a crude concentrate oflipophilic extractables. The crude concentrate was purified bychromatography over lipophilic gel exclusion resins such as SephadexLH-20®(Pharmacia, Piscataway, N.J.) in a solvent such as methanol, or inmixed solvents such as chloroform hexane 1:1. Antibiotic-containingfractions may be detected by bioassay on pH 8 agar media seeded with amacrolide sensitive strain of Staphylococcus aureus.

Separation of antibiotic congeners of similar structure may be achievedby countercurrent chromatography using an Ito Coil Planet Centrifuge®,(P.C., Inc., Potomac, Md.,) with a suitable two-phase system. Multiplechromatographic separations may be required to separate all of thebioactive congeners from a single fermentation.

A 10 liter fermentation of the strain 12693-240 yielded an oil (264 mg)as the crude extractable basic fraction. This was subjected to countercurrent chromatography on an Ito Coil Planet Centrifuge using a 2-phasesystem (n-heptane, benzene, isopropanol, acetone, 0.05 M; pH7.0potassium phosphate buffer in the ratio 5:10:3:2:5, respectively, withthe lower phase as the stationary phase). A flow rate of approximately 4ml/min and a coil spin rate of approximately 800 r.p.m. was maintained.The sample was injected onto the column in 10 ml of upper phase using aRheodyne™ injection loop. Approximately 8 ml fractions were collected.Each fraction was bioassayed against Staphlococcus aureus 6538P using 20μl discs in disc diffusion assay on Streptomycin assay agar plates(Difco) adjusted to pH 8 prior to autoclaving. Fractions were combined,on the basis of bioactivity profile, in three bioactive pools. From eachof these the solvent was removed and the resulting residue waspartitioned between chloroform and dilute aqueous ammonium hydroxide.The chloroform layer was removed, was washed with water, and then wasconcentrated to a residue. These were examined in CDCl₃ by ¹ H-NMRspectroscopy.

Antibiotics were characterized spectroscopically using high yield FT NMRspectroscopy. A variety of one dimensional and two dimensional pulsetechniques were employed with both homo and heteronuclear observationsof ¹ H and ¹³ C nuclei. These data were supplemented by high resolutionmass spectrometry using both fast-atom bombardment and electron impactsources.

A first activity band eluted from the chromatogram was shown to be amixture of closely related macrolides and was rechromatographed.

A second activity band was shown to be 2-norerythromycin D (FIG. 5). Itwas characterized by extensive ¹ H-NMR decoupling experiments and CMRspectrum as respectively illustrated in Tables 1 and 2. In the absenceof published spectra for erythromycin D the comparative data forErythromycins A and B in Table 2 is as published in Terui et al.,Tetrahedron Lett., 1975, 2583-2586 (1975) except that the assignmentsfor 2CH₃ and 8CH³ are interchanged (as was suggested as a possiblyproper assignment in Terui et al., supra). Data for erythromycin C weregenerated at Abbott Laboratories.

In Table 1, TMS is an abbreviation for tetramethylsilane, s is anabbreviation for singlet, d is an abbreviation for doublet, t is anabbreviation for triplet, q is an abbreviation for quartet, and m is anabbreviation for multiplet.

                  TABLE 1                                                         ______________________________________                                        .sup.1 H--NMR SPECTRUM OF 2-NORERYTHROMYCIN D                                         Chemical Shift                                                                              Splitting  Coupling                                     Assigment                                                                             (ppm from TMS)                                                                              pattern    Constants(Hz)                                ______________________________________                                        H-13    5.44          d of d of d                                                                              1.8, 4.2, 9.6                                H-1"    4.99          d (broad)  4.0, <1.0                                    H-1'    4.32          d          7.0                                          H-3     4.24          d of t     11.4, 2.5                                    H-5"    3.84          d of q     9.6, 5.9                                     H-11    3.75          d (broad)  10.0, <1.0                                   H-5'    3.58          d of d of q                                                                              10.7, 1.9, 6.2                               H-5     3.55          d          7.0                                          H-2'    3.25          d of d     7.0, 10.3                                    OH      3.19          s (broad)                                               H-10    3.01          q (broad)  7.0, <1.0                                    H-4"    3.00          d          9.6                                          H-8, H3'                                                                              2.67          m                                                       H-2a    2.66          d of d     12.1, 11.4                                   H-2e    2.50          d of d     3.0, 12.1                                    (CH.sub.3).sub.2 N                                                                    2.37          s                                                       H-4     2.26          q of d of d                                                                              7.0, 7.7, 12.2                               H-7a    2.02          d of d     12.1, ˜15                              H-2" e  2.01          d of d     14.3, <1.0                                   H-2" a  1.88          d of d     14.3, 4.0                                    H-4' e  1.76          d of d of d                                                                              ˜13, 1.5, 1.9                          H-14a   1.71          q of d of d                                                                              1.8, 7.0, ˜13                          H-12    1.65          m                                                       H-7e    1.64          d of d     ˜15, 2                                 H-14b   1.48          q of d of d                                                                              4.2, 7.0, ˜13                          CH.sub.3 --C.sub.6                                                                    1.46          s                                                       CH.sub.3 --6"                                                                         1.33          d          5.9                                          H-4' a  ˜1.30   m                                                       CH.sub.3 --C.sub.3 "                                                                  1.26          s                                                       CH.sub.3 --6'                                                                         1.24          d          6.3                                          CH.sub.3 --C.sub.8                                                                    1.16          d          7.0                                          CH.sub.3 --C.sub.4                                                                    1.12          d          7.7                                          CH.sub.3 --C.sub.10                                                                   1.00          d          7.0                                          CH.sub.3 --15                                                                         0.89          t          7.3                                          CH.sub.3 --C.sub.12                                                                   0.87          d          7.0                                          ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        .sup.13 C--NMR SPECTRUM OF 2-NORERYTHROMYCIN D AND                            COMPARISON WITH VARIOUS ERYTHROMYCINS                                         Assign-                                                                             Erythro- Erythro-  Erythro-                                             ment  mycin A  mycin B   mycin C                                                                              2-Norerythromycin D                           ______________________________________                                        C-1   175.5    175.8     175.5  170.6                                         2     44.8     44.7      45.0   37.6                                          3     80.0     80.2      83.2   78.6                                          4     39.4     39.5      39.4   37.4                                          5     83.6     83.5      85.0   84.0                                          6     74.8     74.8      75.2   75.3                                          7     38.1     38.1      38.6   38.1                                          8     45.8     44.7      45.0   45.5                                          9     221.1    219.1     22.0   220.9                                         10    38.2     39.0      37.8   38.2                                          11    68.7     69.3      69.5   69.5                                          12    74.8     39.8      74.6   39.7                                          13    77.0     75.2      77.3   75.3                                          14    21.3     25.6      21.3   25.6                                          2CH.sub.3                                                                           16.2     15.5      16.2                                                 4CH.sub.3                                                                           9.1      9.2       9.2    8.5                                           6CH.sub.3                                                                           26.8     27.3      26.9   27.2                                          8CH.sub.3                                                                           18.5     18.5      18.4   18.0                                          10CH.sub.3                                                                          12.0     9.2       11.9   9.0                                           12CH.sub.3                                                                          16.0     9.2       16.0   9.5                                           14CH.sub.3                                                                          10.5     10.3      10.6   10.4                                          1'    103.1    103.0     104.8  103.6                                         2'    70.9     70.9      70.8   70.6                                          3'    65.3     65.5      65.5   65.5                                          4'    28.7     28.7      28.4   28.9                                          5'    68.8     68.8      68.8   69.2                                          6'    21.3     21.3      21.0   21.3                                          N(CH.sub.3).sub.2                                                                   40.3     40.2      40.2   40.2                                          1"    96.2     96.5      98.7   94.9                                          2"    35.0     35.0      40.4   41.4                                          3"    72.5     72.5      69.4   69.7                                          4"    77.9     77.9      76.4   76.4                                          5"    65.4     65.6      66.1   66.6                                          6"    18.3     18.5      18.3   18.4                                          3"CH.sub.3                                                                          21.3     21.3      25.5   25.7                                          OCH.sub.3                                                                           49.4     49.4                                                           ______________________________________                                    

In Table 1, the splitting pattern of a doublet of a triplet for H-3indicates that H-3 is coupled to three other protons which have to beprotons on C-2 or C-4, so that a methyl group must be missing from thematerial in the second activity band. The splitting pattern for H-5 is asharp doublet indicating that no extra proton is present on C-4.Furthermore, as shown in Table 2, the absence of methyl substitution atC-2 is consistent with the change in the ¹³ C spectrum of the positionof the peak from the position it occupies for erythromycins A, B, and C.

The third activity band was shown to be 2-norerythromycin C, illustratedin FIG. 3, by an ¹ H-NMR spectrum, as indicated in Table 3, and by highresolution mass spectrum. The third activity band exhibited an FABpositive ion MH⁺ m/z of 706.

In Table 3, TMS is an abbreviation for tetramethylsilane, s is anabbreviation for singlet, d is an abbreviation for doublet, t is anabbreviation for triplet, q is an abbreviation for quartet, and m is anabbreviation for multiplet.

                  TABLE 3                                                         ______________________________________                                        PARTIAL .sup.1 H--NMR SPECTRUM OF                                             2-NORERYTHROMYCIN C                                                                                            Approximate                                             Chemical Shift                                                                             Splitting                                                                              Coupling                                     Assignment (ppm from TMS)                                                                             Pattern  Constants (Hz)                               ______________________________________                                        H-13       5.10         d of d   2.7, 11                                      H-1"       5.0          d (broad)                                                                              3, <1                                        H-1'       4.28         d        7                                            H-3        4.22         d of t   9.6, ˜2                                H-5"       3.8          m                                                     H-11       3.75         s (broad)                                             H-5        3.54         d        7.2                                          H-5        3.53         m                                                     H-2        3.21         d of d   7.0, 10.5                                    H-10       3.1          q broad  7.2                                          H-4"       3.0          d        9.3                                          H-2a       2.68         t        11.8                                         H-8        2.65         m                                                     H-3        2.60         m                                                     H-2e       2.46         d of d   2.6, 11.8                                    (CH.sub.3).sub.2 N                                                                       2.3          s                                                     H-4        2.13         broad    7.2                                                                  quintet                                               CH.sub.3 --6                                                                             1.48         s                                                     CH.sub.3 --6"                                                                            1.33         d        6.0                                          CH.sub.3 --C.sub.12,                                                                     1.26         s                                                     CH.sub.3 --C.sub.3 "                                                          CH.sub.3 --15                                                                            0.85         t        7.2                                          ______________________________________                                    

In Table 3, the position of the splitting pattern of the signal at 4.22,readily assignable to H-3, is indicative in this molecule that a methylgroup is missing from the C-2 position. The coupling pattern of protonsignal of a doublet of doublets at 5.10 assigned to the H-13 proton isindicative of hydroxylation of C-12 of the macrolide structure as inerythromycins A and C. This, in conjunction with the mass spectral data,supports assignment of the third activity band as 2-norerythromycin C.

The first activity band was rechromatographed on an Ito Coil PlanetCentrifuge® using as the two-phase system carbon tetrachloride,choloroform, methanol, 0.01 M pH 5 aqueous sodium acetate-acetic acidbuffer in the ratio 1:1:1:1 with the lower phase as the stationaryphase. The flow rate was approximately 4 ml/min., the coil spin rate of800 r.p.m. and the injection volume 2 ml. Three activity bands wereeluted. A first activity band eluted from the column was identified by ¹H-NMR as 2-norerythromycin A as ¹ illustrated in FIG. 3. Mass spectrum,FAB positive ion MH⁺ m/z was 720.

The second activity band was identified by ¹ H-NMR spectrum aserythromycin A (identical to the spectrum of an authentic sample).

The third activity band was identified as 2-norerythromycin B asillustrated in FIG. 5 by ¹ H-NMR spectrum and by mass spectrum FABpositive ion MH⁺ m/z of 704.

Mass spectra were run in the fast atom bombardment (FAB) positive ionmode on the first and third activity bands of the rechromatographedmaterial, as noted above. The peaks attributable to the protonatedmolecular ion (MH+) species observed at m/z 720 and m/z 704 wererespectively consistent with mass/charge (m/z) ratios expected for2-norerythromycin A and 2-norerythromycin B. In each case, a majordegradation ion peak attributable to the protonated basic fractionresulting from loss of a neutral sugar from the corresponding parent wasobserved. This peak was at m/z 562 for 2-norerythromycin A and m/z 546for 2norerythromycin B. These mass spectral peaks and their associationwith bioactive bands co-produced with wellcharacterized2-norerythromycins C and D provide strong circumstantial evidence for anidentification of the bioactive components as being 2-norerythromycin Aand 2norerythromycin B.

It is well known that erythromycin A in its pharmaceutically useful formis subject to acidic degradation to 8,9-anhydroerythromycinA-6,9-hemiketal and to erythromycin A-6,9,12 spiroketal. Both of theseproducts are essentially devoid of antibacterial activity. Intermediatesin the formation of these inactive products are the various possibleepimeric hemiketals involving the 6 or 12 hydroxyl groups and the9-ketone function. These four possible hemiketals are normally intautomeric equilibrium with the dihydroxy ketone form. It is interestingthat an examination of chemical shift differences between the carbons of2-norerythromycin D and appropriate carbons from available referencespectra of erythromycin B and erythromycin C, as provided in FIG. 4,shows three specific areas of major difference. In FIG. 4, differencesbetween spectral values for 2-norerythromycin D and the indicatederythromycin reference are given adjacent the position in theerythromycin structure to which they correspond. The largest differencesoccur in the region of C-2, as would be expected for change orsubstitution at that position. Major differences also occur in thevicinity of the anomeric carbons of the sugars. This probably reflects alesser degree of conformational rigidity for these sugars in the 2-norcompound as compared to the parent antibiotics. The other postion ofmajor difference is in the vicinity of C-9 and this most probablyreflects differences in the relative tautomeric populations of thehemiketal forms of 2-nor compounds as compared to those for the 2-methylparents. This difference will almost certainly result in differences inrates of conversion to the 8,9 anhydro-6,9 hemiketal and the 6,9,12spiroketal derivatives (where applicable) between 2-norerythromycins andcurrently available erythromycins, and, hence, an increase in thestability of the 2-norerythromycins over the currently availableerythromycin antibiotics.

In addition, the quantity of material assayed with the size of zonessurrounding discs in the disc assay described above indicate that2-norerythromycin A and 2norerythromycin B are potent antibiotics, atleast comparable in potency to the corresponding erythromycin A and B.Similarly, although 2-norerythromycin C and 2norerythromycin D aresuggested to be relatively weaker antibiotics than 2-norerythromycins Aand B, this relative potency is consistent with the relative potency ofthe corresponding erythromycins C and D which are relatively weakerantibiotics than are erythromycins A and B.

Although the present invention has been described in terms of apreferred embodiment, it is understood that variations and modificationswill occur to those skilled in the art. For example, although plasmidspAL7002 and pNJl employ a ˜2.7 kb SmaI restriction fragment of pJVl as afunctional replication segment in Steptomyces, other segments of pJVlmay be used, including a ˜4.6 kb BamHI fragment of a ˜4.7 kb KpnIfragment. These fragments are ligated to a segment of DNA containing thegenetic determinant for thiostrepton resistance in Streptomyces. Inaddition although in the present invention, the segment of DNAdetermining thiostrepton resistance described herein was a ˜1.05 Kb BClIfragment, obtained from the plasmid pIJ704, other fragments containingthe thiostrepton resistance gene may also be employed, including all orpart of plasmids pIJ350, pI702, pI703, pI704, pI705, or pIJ61 asexamples, all of which may be obtained from the collection at the JohnInnes Institute, Norwich, U.K.. Plasmid pIJ702, available from theAmerican Type Culture Collection, Rockville, Md., under the accessionnumber 35287, may be directly substituted in the procedures employingpIJ704 herein. Alternatively, the thiostrepton resistance gene may beisolated from the original source, the genome of the strain Streptomycesazureus and ligated to the functional replication segment of pNJl orpAL7002.

Likewise, although DNA segments conferring resistance to thiostreptonwas exemplified herein, other DNA segments which confer resistance tothe same or different antibiotics, including erythromycin, streptomycin,chloramphenicol, hygromycin, neomycin, tylosin, picromycin and the likecan be used by those skilled in the art either as replacements of, or inaddition to the drug resistance segments described here, by ligation tothe replication-determining segments of pAL7002 or pNJl to result infunctional cloning vectors that are within the scope of the presentinvention.

While various restriction fragments were ligated together to formplasmids pAL7002 and the other vectors of the present invention, thoseskilled in the art understand that it is possible to modify thefragments to facilitate ligation by either attaching DNA oligonucleotidelinkers to the fragments or by creating blunt ends on the fragments by avariety of techniques to allow non-homologous ends to be ligatedtogether or to permit the construction of novel restriction sites ofpAL7002 or its derivatives or the other vectors present in the inventionto facilitate subsequent cloning experiments. Furthermore, while thevectors of the present invention represent the products of ligation ofvarious restriction fragments to yield a given DNA sequence evidenced bythe restriction maps shown for the present invention, those skilled inthe art understand that ligation of two fragments with homologousrestriction-cut ends will yield products with two possible orientationsbut which share all the properties of the vectors described for thepresent invention. The present invention, therefore, includes within itsscope, all functional vectors constructed from the same restrictionfragments as those described here but which are arranged within thevector in any of all possible orientations.

The cloning vectors present in this invention are not limited for use ina single species or strain of Streptomyces but are applicable to a widevariety of species and strains, especially those which produceantibiotics or other compounds which may be converted into antibioticsby chemical on biological means. Such species or strains which produceantibiotics of the macrolide type include, but are not limited to S.erythreus, S. antibioticus, S. venezuelae, S. fradiae, and S.narbonensis, as examples. Such species or strains which produceantibiotics of the aminoglycoside type include, but are not limited toS. kanamyceticus, S. tennebrarius, S. hygroscopicus, S. kasugaenis, andS. bikiniensis, as examples. Such strains or species which produceantibiotics of the β-lactam type include, but are not limited to S.clavuligerus, S. lactamdurans, S. viridochromogenese, S. rochei, and S.flavus, as examples. Such strains or species which produce antibioticsof the polyether type include but are not limited to S. albus, S.griseus, S. aureofasciens, S. ribosidificus, and S. violaceoniger, asexamples. Such specie or strains which produce antibiotics of theglycopeptide type include, but are not limited to S. orientalis and S.candidus, as example. The vectors of the present invention are alsoparticularly useful in other hosts of Streptomyces including, but notlimited to S. lividans 66, S. coelicolor, and S. parvulus, as examples,which do not fall into the categories listed above.

Although methods for the transformation of protoplasts of S. lividansand S. erythreus are described in the present invention, those skilledin the art understand the methodology employed for the generation ofprotoplasts of Streptomyces and transformation of said protoplasts withthe vectors of the present invention, including the use of regenerationmedium and selection for antibiotic resistance carried on the vectorsmay be applied to transformation of other strains.

Accordingly, it is intended that all such equivalent modification andvariations should come within the scope of the invention as claimed.

We claim:
 1. The compound 2-norerythromycin A, which is a compound of the formula: ##STR1## or a pharmaceutically acceptable salt thereof.
 2. 2-norerythromycin A as recited in claim 1 wherein said 2norerythromycin A in substantially pure form has a mass spectrum, FAB ion MH⁺ m/z of
 720. 3. The compound 2-norerythromycin B, which is a compound of the formula: ##STR2##
 4. 2-norerythromycin B as recited in claim 3 wherein said 2-norerythromycin B in substantially pure form has a mass spectrum, FAB positive ion MH⁺ m/z of
 704. 5. The compound 2-norerythromycin C, which is a compound of the formula: ##STR3## or a pharmaceutically acceptable salt thereof.
 6. 2-norerythromycin C as recited in claim 5 wherein said 2-norerythromycin C in substantially pure form has an FAB positive ion MH+ m/z of 706 and has an ¹ H-NMR spectrum comprising the partial spectrum as set forth in Table
 3. 7. The compound 2-norerythromycin D, which is a compound of the formula: ##STR4## or a pharmaceutically acceptable salt thereof.
 8. 2-norerythromycin D as recited in claim 7 wherein said 2-norerythromycin D in substantially pure form has an ¹ H-NMR spectrum as set forth in Table 1 and has a ¹³ C-NMR spectrum as set forth in Table
 2. 9. An antibiotic composition comprising:a diluent or carrier compatible with antibiotic activity; and an effective amount of an antibiotic selected from the group consisting of:2-norerythromycin A: ##STR5## or a pharmaceutically acceptable salt thereof; 2-norerythromycin B: ##STR6## or a pharmaceutically acceptable salt thereof; 2-norerythromycin C: ##STR7## or a pharmaceutically acceptable salt thereof; and 2-norerythromycin D: ##STR8## or a pharmaceutically acceptable salt thereof. 